The present invention is directed to transgenic non-human animals in which a cAMP phosphodiesterase is selectively inactivated. In particular, the non-human transgenic animals of the present invention contain a disrupted PDE7A gene, which results in abrogation of expression of wild-type PDE7A-encoded proteins. The transgenic animals of the present invention may exhibit reduced or no expression of PDE7A gene products, expression of mutant PDE7A gene products, or expression of only human PDE7A gene products (wild-type or mutant). The present invention is also directed to methods for making the transgenic animals of the present invention. Finally, the present invention is directed to methods for using these transgenic animals to screen therapeutic compounds and to modulate immune and other physiological responses.
Cyclic AMP (cAMP) is a second messenger with broad physiological implications, including anti-proliferative effects in lymphoid and myeloid cells, endocrine cells, mammary cells, and prostate cells. In addition, reduced cAMP levels are associated with certain cancers (e.g., breast tumors, chronic lymphocytic leukemia (CLL), and prostate tumors), which may be treated with cAMP elevating agents.
Phosphodiesterases (PDEs) are enzymes, present in cells, that are important regulators of immune function. Lymphoid and myeloid cells contain multiple cyclic nucleotide PDEs that lower cAMP levels in response to antigenic stimulation, thereby promoting cell proliferation. For example, phophodiesterase 7A (PDE7A) is proposed to be a critical regulator of lymphocyte proliferation, although in vivo data in this regard are lacking. PDE3, PDE4, and PDE7A are present in T cells. Each belongs to a different PDE family. These PDE families are related to each other, in that they share a 25% sequence similarity within their catalytic domains, but their regulatory domains are distinct. Selective inhibitors exist for the catalytic domains of particular PDE families. For example, sildenafil (Viagra(trademark)), an inhibitor of PDE5, is marketed for the treatment of erectile dysfunction. Selective inhibitors for PDE4 are immunosuppressant drugs in different stages of development. However, the wide tissue distribution of PDE4 isozymes leads to side-effects of PDE4 inhibitors, such as emesis. PDEs that are expressed in a tissue-selective manner, and that respond to specific-activating signals, are good targets for elevation of cAMP in specific physiological conditions.
Immunologic responses and immunodeficiency conditions (e.g., allergy; asthma; autoimmune disorders, such as Hashimoto""s thyroiditis, systemic lupus erythematosus (SLE), Goodpasture""s syndrome, Crohn""s disease, pemphigus, receptor autoimmunity, Graves"" disease, myasthenia gravis, insulin resistance, and autoimmune hemolytic anemia; chronic obstructive pulmonary disease (COPD); CLL; inflammation; and rheumatoid arthritis) are either caused by, or associated with, proliferation and/or accumulation of lymphoid cells. Inhibition of cAMP PDEs in lymphoid cells, though, can inhibit the antigen-stimulated proliferation of these cells. In particular, since PDE7A is a primary modulator of lymphocyte proliferation, it is believed to play a role in immunologic responses and immunodeficiency conditions such as those described above.
Phosphodiesterase inhibitors are currently being investigated for their potential to modulate immune function. For example, inhibitors have been developed which target PDE4xe2x80x94a phosphodiesterase known to affect lymphocyte proliferation. However, PDE4 inhibitors have been shown to have deleterious side-effects, such as severe emesis, that preclude their use clinically. Because of PDE7A""s apparent role in immunologic responses and immunodeficiency diseases, therapeutic strategies designed to inhibit PDE7A activity (by decreasing PDE7A levels and/or by reducing PDE7A activity) also have been proposed.
Current mice models for immunodeficiency diseases and for immune responses to allergens, antigens, superantigens, and transplanted tissues all express PDE7A. Furthermore, to date, the study of PDE7A function, particularly in muscle, has been hindered by the absence of selective inhibitors for this phosphodiesterase, the absence of cultured cell lines expressing the muscle PDE7A2 splice variant (including cultured myoblast cell lines like C2C12), and the absence of cell lines that can serve as bona fide physiological models for muscle function. Accordingly, the development of viable PDE7-knockout (PDE7KO) mice would allow a physiological determination of defects in PDE7A function, provide a source of control tissues for comparative analysis of PDE7A activity, and facilitate the identification of PDE7A inhibitors and the evaluation of their efficacy.
The present invention is directed to the development of novel transgenic non-human animals in which a cAMP phosphodiesterase has been selectively inactivated. Specifically, the inventor has produced a genetically-engineered mouse in which one of the PDEs, PDE7A, has been knocked out. This mouse expresses a reduced level of PDE7A-encoded proteins relative to the corresponding wild-type animal. The mouse also has significant immune defects that appear to be distinct from those seen in other PDE-knockout mice.
The transgenic mouse of the present invention will allow researchers to use convenient and well-studied small laboratory animals, including rats or mice, to study immune responses and other conditions and conditions associated with lymphoid cell proliferation. Because the transgenic mouse does not express endogenous PDE7A, it also can be used to study the long-term sequellae induced by treatment with drugs that inhibit PDE7A. Therefore, the invention will be useful for testing side-effects of compounds that inhibit PDE7A, and for screening candidate therapeutic compounds that block the proliferation of lymphoid and other immune cells.
Accordingly, the present invention provides a transgenic non-human animal whose genome comprises a disruption in its endogenous PDE7A gene, wherein the transgenic animal exhibits decreased expression of functional PDE7A protein relative to wild-type.
In addition, the present invention provides a method for creating a transgenic non-human animal exhibiting decreased expression of functional PDE7A protein relative to wild-type by: (a) generating a PDE7A targeting vector; (b) introducing the PDE7A targeting vector into a recipient cell of a non-human animal, to produce a treated recipient cell; (c) introducing the treated recipient cell into a blastocyst of a non-human animal, to produce a treated blastocyst; (d) introducing the treated blastocyst into a pseudopregnant non-human animal; (e) allowing the transplanted blastocyst to develop to term; (f) identifying a transgenic non-human animal whose genome comprises a disruption in its endogenous PDE7A gene; and (g) breeding the transgenic non-human animal to obtain a transgenic non-human animal exhibiting decreased expression of functional PDE7A protein relative to wild-type.
Finally, the present invention provides a method for screening a PDE7A inhibitor for at least one side-effect, by administering a PDE7A inhibitor to the transgenic non-human animal of the present invention, and detecting at least one side-effect, if any, of the PDE7A inhibitor in the transgenic animal.
Additional objects of the present invention will be apparent in view of the description which follows.